This invention relates to a new technique permitting rationalization and automation of microbiological, serological, immunological, clinical-chemical and similar laboratory work.
Before the invention is explained, it will be expedient to give some general background information.
Bacteria are grown in substrates (media) which are either liquid (broth) or of more or less solid consistency. Growth in liquid substrates takes place throughout the entire substrate, while in solid substrates the growth usually takes place on the surface.
The solid substrates are made from the liquid by adding agar (or some other substance, such as gelatine) in various concentrations, or through the coagulation of albumin. There are a number of different types of liquid and solid substrates. Often they have a common basic composition, but otherwise the composition changes according to which substances are required for the growth of the bacteria in question.
Many, though not all, bacteria decompose and utilize matter in different ways. This can be registered by various chemical or physico-chemical reactions. As an example may be mentioned the fermentation or splitting of various types of so-called "sugar" (hereinafter called sugar) by bacteria. This fermentation, considered for the whole series of sugar types in question, is often characteristic of the individual type of bacterium, and frequently differs from one bacterium to the other. This circumstance is widely exploited as an aid in distinguishing bacteria from one another.
When a bacterium decomposes a sugar to acid products, the pH value in the substrate decreases, as may be observed by a change in the color of an added indicator. If, for example, a sugar is added to a solid substrate as a fermentation basis, and bromothymol blue is added as an indicator, the result of inoculation with bacteria that break down sugar, and with bacteria that do not, is as follows: The sugar-splitting bacteria will grow on the surface of the substrate and form yellow colonies, while the non-splitting bacteria will form colonies of the same blue color as that of the substrate before inoculation. Yellow and blue colonies may lie close together, side by side, on the surface of the substrate.
A corresponding liquid substrate will change its color to yellow if sugar-splitting bacteria grow in it, and will remain blue if the growth is purely of non-splitting bacteria.
When, as an aid to classification, fermentation determinations are carried out, liquid substrates are usually employed.
Since one of the objects of the invention is to rationalize fermentation tests--without limiting its scope to this field--a more detailed description will be given of how a fermentation test is carried out according to a known method.
A pure culture of the micro-organism to be classified by the fermentation test must be prepared. Samples from this pure culture are inoculated into broths which have already been poured into test tubes. These broths contain an indicator and various types of sugar. The test tubes are closed with plugs or caps. The test tubes containing the broths are placed in racks and incubated for growth and reaction. The result is read by inspecting each tube, and is noted by hand.
This method requires that the test tubes be placed in the racks by hand. Each tube of broth contains only one kind of sugar, but the number of kinds of sugar, and thus of tubes, may often be comparatively high. This means that handling the tubes and segregating them according to type of sugar when they are to be arranged in rows in the racks, can be a relatively laborious process. Furthermore, there is room for confusion and error.
The inoculation process is also relatively laborious. The inoculation of each test tube containing the sugar broth involves removal of the stopper, the upper edge of the tube being sterilized by a flame to remove any possible microbial contamination, and the stopper being replaced.
The test tubes, which are often of glass, must, after having been read, go through a time-consuming and laborious process before they can be used anew. First they are put in an autoclave to kill the cultures. Then they are empited, washed, and rinsed, whereupon they are fitted with stoppers. After this they are sterilized and are once again ready to receive new charges of broths containing various types of sugar and indicator.
The preparation of the substrates to be put into the test tubes also demands time and labor. First, the broth must be prepared and then, to portions of this, a number of different types of sugar have to be added. These mixtures are then poured into test tubes and duly sterilized. The tubes are marked to indicate the type of sugar therein. They are then placed in a refrigerator, ready for use.
The test tubes require a good deal of space on account of their relatively large size and number. This makes itself felt in the laboratory work, during storing in the refrigerator and incubator, storing and transport from producer to user. In addition, the racks require storing and cleaning.
The fermentation test described above can also be carried out by positioning prefabricated bodies carrying the various types of sugar in a known solid substrate in a dish or container. The indicator may be conveniently present in the sugar bodies, or, alternatively, it may, in known manner, have been added to the substrate in advance.
Before the bodies are positioned, the substrate surface in the dish is evenly inoculated all over with a suspension or broth containing a pure culture of the micro-organism.
After the bodies have been introduced onto the surface of the substate, the sugar--and the indicator if this has been added to the bodies--will diffuse out into the substrate. This takes place relatively fast in the substrates normally used. As a result, a successful reaction will not occur if the sugar is applied via a paper disc into which it has been absorbed, or via a tablet into which the sugar has been mixed. The diffusion must be delayed or limited.